A vast majority of biologically active molecules including growth factors, cytokines, neurotransmitters and hormones transduce signals via specific cell-surface receptors. Some of these receptors are then coupled to heterotrimeric GTP-binding proteins (G proteins) which, upon activation, relay signals to a variety of cellular effectors including at least four phospholipase C (PLC) variants and adenylyl cyclases.
G proteins mediate external signals by forming heterotrimers consisting of an alpha, beta and gamma subunit. Several isoforms of each subunit have been identified and therefore, through subunit heterogeneity, G proteins effectively integrate multiple signaling cascades. The alpha subunits of G proteins contain the GTP binding site and intrinsic catalytic GTPase activity. Based on sequence similarity and function, these subunits have been classified into four major groups; Gs, which stimulate adenylyl cyclases; Gi, which inhibit adenylyl cyclases; Gq, which activate PLC isoforms and G12/13, which mediate pathways associated with cell growth and differentiation (Hamm, J. Biol. Chem., 1998, 273, 669-672).
G-alpha-12 is a member of the G12/13 subfamily of G proteins and is activated by a variety of stimuli. It has been shown to participate in several kinase cascades including MAPK, ERK and JNK (Jho et al., J. Biol. Chem., 1997, 272, 24468-24474; Voyno-Yasenetskaya et al., J. Biol. Chem., 1996, 271, 21081-21087) as well as being phosphorylated by protein kinase C (Kozasa and Gilman, J. Biol. Chem., 1996, 271, 12562-12567). Consequently, G-alpha-12 has been shown to participate in cellular processes as diverse as mitogenesis (Mitsui et al., J. Biol. Chem., 1997, 272, 4904-4910), regulation of pH (Lin et al., J. Biol. Chem., 1996, 271, 22604-22610) and platelet activation (Collins et al., Oncogene, 1997, 15, 595-600).
The primary function of G-alpha-12, however, is believed to be the mediation of cell differentiation and growth. Therefore the manifestations of altered G-alpha-12 expression, specifically as it relates to the development of cancer, have been the most broadly investigated.
Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer and these processes are believed to be linked to the expression of G-alpha-12.
G-alpha-12 was first isolated from a soft tissue sarcoma cDNA library and overexpression of the protein was shown to be sufficient to induce cellular transformation (Chan et al., Mol. Cell. Biol., 1993, 13, 762-768).
In studies of NIH3T3 and Rat-1 fibroblasts, constitutively active mutant forms of G-alpha-12 were shown to induce transformation as indicated by increased proliferation, loss of contact inhibition, focus formation and increased DNA synthesis (Voyno-Yasenetskaya et al., Oncogene, 1994, 9, 2559-2565).
Results from studies in human embryonic kidney cells demonstrated that constitutive activation of G-alpha-12 stimulated RhoA-dependant phosphorylation of p125 focal adhesion kinase, paxillin and p130 Crk-associated substrate, all of which have been implicated in the regulation of proliferation and transformation (Needham and Rozengurt, J. Biol. Chem., 1998, 273, 14626-14632).
Finally, studies using both antisense vectors expressing a 43 base fragment of mouse G-alpha-12 in antisense orientation and constitutively active forms of G-alpha-12 to investigate retinoic acid-stimulated differentiation of P19 mouse embryonal carcinoma cells found that G-alpha-12 activity was essential to the differentiation process (Jho et al., J. Biol. Chem., 1997, 272, 24468-24474; Jho and Malbon, J. Biol. Chem., 1997, 272, 24461-24467).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of G-alpha-12 and strategies aimed at inhibiting or investigating G-alpha-12 function have involved the use of antibodies, mutant forms of the protein which are constitutively active and antisense expression vectors.
These strategies are untested as therapeutic protocols and consequently there remains a long felt need for additional agents capable of effectively inhibiting G-alpha-12 function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of G-alpha-12 expression.